Palmitoylation is a well-recognized post-translational lipid modification important for protein-membrane association, protein-protein interaction, and protein stability. Intracellular protein palmitoylation is carried out by a newly described family f protein S-acyl transferases (PATs), referred to as the DHHC family, named for a common catalytic motif. The family is comprised of 23 members in humans and the role of these in cancer is just beginning to be explored. In a recent genome-wide screen conducted at UT Southwestern, and a more targeted screen performed in the Hamon Center for Therapeutic Oncology research, the DHHC5 gene was found to contribute to the growth of non-small lung cancer cell lines and tumor xenografts. The goal of this proposal is to gain a better understanding of the mechanism(s) whereby DHHC5 contributes to tumor growth and to develop assays that will serve as the basis for development of inhibitors. Two specific aims are proposed. In Specific Aim 1 we will determine the basis for selective vulnerability of NSCLCs to DHHC5 knockdown. We will test the hypothesis that DHHC knockdown inhibits cell growth and tumor xenograft formation though inhibition of palmitoylation of substrates important for cancer maintenance. To test this hypothesis, we will identify further cell lines sensitive and resistant t DHHC5 knockdown and identify potential pathways for DHHC5 addiction, drawing upon the extensive database available at UT Southwestern. We will identify candidate substrates by profiling palmitoylation of proteins in control and DHHC5 knockdown cell lines, using methods we have previously published. Candidate DHHC5-substrate pairs will be validated through expression assays involving heterologous cells, and then evaluated as targets in lung cancer cell lines using appropriately designed siRNA and overexpression experiments. In Specific Aim 2, we will determine structural features of DHHC5 needed to support growth of NSCLC lines and tumors. We have identified flotillin-2 as a physiological substrate of DHHC5 in neuronal cells, and it is a candidate substrate in NSCLC. Using flotillin-2 as a model substrate for DHHC5, we aim to learn about residues involved in catalysis and substrate recognition that would support the eventual development of inhibitors. These studies may be of significant value to the 160,000 individuals in the US who will succumb to lung cancer annually.